Gene expression in tonsil and oral epithelia. Homo sapiens

To study characteristics of the orapharyngeal epithelia which may influence susceptibility or resistance to HIV, we performed microarray analysis of the tonsil and gingival epithelium.Tonsil epithelium has been implicated in HIV pathogenesis, but its role in oral transmission remains controversial. We performed microarray analysis of Laser Capture Microdissected tonsil and gingival epithelium. Our data revealed that genes related to immune functions such as antibody production and antigen processing were increasingly expressed in tonsil compared to the epithelium of another oro-pharyngeal site, gingival epithelium. Importantly, tonsil epithelium highly expressed genes associated with HIV entrapment and/or transmission, including the HIV co-receptor CXCR4 and the potential HIV binding molecules, FcRγIII, complement receptor 2, and various complement components. This increased expression of molecules involved in viral recognition, binding and entry may favor virus-epithelium interaction in an environment with reduced innate anti-viral mechanisms. Specifically, secretory leukocyte protease inhibitor, an innate molecule with anti-HIV activity, was minimal in the tonsil epithelium, in contrast to oral mucosa. Collectively, our data suggest that increased expression of molecules associated with HIV binding and entry coupled with decreased innate anti-viral factors may render the tonsil a potential site for oral transmission. Keywords: Cross sectional Overall design: To study characteristics of the orapharyngeal epithelia which may influence susceptibility or resistance to HIV, we performed microarray analysis of the tonsil and gingival epithelium. Human palatine tonsils were obtained from routine therapeutic tonsillectomies (sleep apnea and non-tonsillitis) performed on otherwise healthy adults at the George Washington University Hospital with informed consent (IRB #099920). Gingival tissues were collected from healthy sites with probing depths < 3mm, with no clinical evidence of inflammation during routine therapeutic periodontal surgery at the University of Maryland, Department of Periodontics, with informed consent (IRB#1201211). Tissues were immediately snap frozen for the microarray studies. CM performed immediately at 10x magnification, using a Leica AS LMD system (Leica Microsystems). Areas unequivocally identified as epithelium were outlined , laser-dissected and captured and RNA from the tissues isolated. Preparation of biotin-labeled cRNA, hybridization, and scanning were performed according to manufacturer’s two cycle protocol (Affymetrix, Santa Clara, CA). Fluorescence intensity was measured using the Affymetrix GeneChip scanner and GeneChip Operating Software (GCOS, Affymetrix). Samples GSM173679, GSM173680,GSM173681, GSM173682, GSM173683, GSM173684, GSM173685, GSM173686, GSM173688, GSM173690 were processed togehter as "batch 1", while samples GSM173687 and GSM173689 were processed as "batch 2" together with sample GSM173691 which is a repeat of GSM173690, which was processed for comparison between the two batches.

为探究可能影响人类免疫缺陷病毒（HIV）易感性或抗性的口咽上皮特征，我们对扁桃体及牙龈上皮开展了基因芯片分析。扁桃体上皮已被证实与HIV发病机制相关，但其在口腔传播中的作用仍存在争议。我们对经激光捕获显微切割（Laser Capture Microdissection, LCM）的扁桃体及牙龈上皮开展了基因芯片分析。

本研究数据显示，与另一口咽部位点的上皮——牙龈上皮相比，扁桃体上皮中与免疫功能（如抗体生成、抗原加工）相关的基因表达水平显著上调。尤为关键的是，扁桃体上皮高表达与HIV捕获和/或传播相关的基因，包括HIV共受体CXCR4，以及潜在的HIV结合分子FcRγIII、补体受体2与多种补体成分。这种参与病毒识别、结合与入侵的分子表达上调，可能在先天抗病毒机制减弱的微环境中，促进病毒与上皮的相互作用。

具体而言，分泌性白细胞蛋白酶抑制剂（secretory leukocyte protease inhibitor, SLPI）——一种具有抗HIV活性的先天免疫分子——在扁桃体上皮中的表达量极低，这与口腔黏膜形成鲜明对比。

综上，本研究数据表明，与HIV结合、入侵相关的分子表达上调，加之先天抗病毒因子表达下调，可能使扁桃体成为HIV口腔传播的潜在位点。

关键词：横断面研究
整体实验设计：
为探究可能影响HIV易感性或抗性的口咽上皮特征，我们对扁桃体及牙龈上皮开展了基因芯片分析。人腭扁桃体样本取自乔治华盛顿大学医院对健康成人开展的常规治疗性扁桃体切除术（针对睡眠呼吸暂停及非扁桃体炎患者），所有受试者均签署知情同意书（伦理审查编号IRB #099920）。牙龈组织取自马里兰大学牙周病科常规治疗性牙周手术中探诊深度<3mm的健康位点，无临床炎症证据，受试者均签署知情同意书（伦理审查编号IRB#1201211）。

组织样本获取后立即置于液氮中快速冷冻，用于后续基因芯片研究。激光捕获显微切割操作在10倍放大倍率下进行，采用Leica AS LMD系统（徕卡显微系统公司）。明确判定为上皮的区域被勾勒出轮廓，经激光切割捕获后，提取组织中的总RNA。

生物素标记的互补RNA（cRNA）制备、杂交与扫描流程均按照制造商的双循环实验方案执行（Affymetrix，美国加利福尼亚州圣克拉拉市）。荧光信号强度采用Affymetrix GeneChip扫描仪与GeneChip操作软件（GCOS，Affymetrix）进行检测。

样本GSM173679、GSM173680、GSM173681、GSM173682、GSM173683、GSM173684、GSM173685、GSM173686、GSM173688、GSM173690作为“批次1”统一处理；样本GSM173687、GSM173689与作为GSM173690重复样本的GSM173691一同作为“批次2”处理，用于对比两个批次间的实验差异。